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Viruses 2016, 8, 300 2 of 11
Pithovirus massiliensis, from sewage, offered the opportunity to analyze the evolution between the
modern and fossil genomes [9]. Surprisingly, the estimated mutation rate was found to be lower
than RNA viruses and some DNA viruses, and these genomes exhibited stability comparable to that
of prokaryotic genomes. Morphologically, previously described pithoviruses have a single cork at
the apex region, suggesting a polarity essential for the infection of amoebas [7]. We describe herein
a new giant virus isolate, morphologically and genetically close to pithoviruses, which was named
Cedratvirus because of its lemon-shape (Citrus medica). Cedratvirus has an ovoid virion and a cork
at each apex. Genomic analyses revealed that Cedratvirus is most closely related to pithoviruses
and may expand the putative Pithoviridae family, but displays new features, such as a different set of
genes involved in the biosynthesis of sugars and amino-acids, and a distant relationship with the two
previously described pithoviruses.
2. Materials and Methods
2.1. Culture and Isolation Procedures
Twenty-four samples were collected in October 2015 from Constantine, Annaba, Taref, El-Kala and
Oran, in Algeria. We performed a classic co-culture method associated with presumptive identiﬁcation
by ﬂow cytometry [9,10]. Acanthamoeba castellanii strain Neff was used as cell support. The amoebas
were harvested after 48 h in culture in Peptone Yeast Extract Glucose medium (PYG, home made) when
they reached a concentration of 5.105 amoebas/mL. Cells were rinsed twice in Page’s Amoeba Saline
(PAS, home made) and pelleted at 700× g for 10 min. Afterwards, the amoebas were re-suspended in
the starvation medium [10] at a concentration of 5.105 amoebas/mL. Antibiotic and antifungal mixture
with vancomycin (10 µg/mL), ciproﬂoxacin (20 µg/mL), imipenem (10 µg/mL) and voriconazole
(20 µg/mL) were added to the suspension in order to decrease or eliminate bacterial or fungal
contaminations. Cell suspension was then distributed in a 48-well plate at the level of 250 µL per well,
the samples were vortexed and 50 µL were added to each well. The rest of the wells served as negative
control by adding 50 µL of PAS. The plate was incubated at 30◦C for four days. The cytopathic effect
was monitored under optical inverted microscope. This co-culture was repeated twice in the same
order. This technique is well detailed and discussed in the work of Bou Khalil et al. [11].
2.2. End-Point Dilution and Centrifugation Methods
In order to obtain a pure viral population, viral supernatant was centrifuged at 4000× g for 15 min.
The pellet was re-suspended with 1 mL of PAS three times. Finally, fresh A. castellanii cells were
inoculated with the re-suspended pellet.
End-point dilution was performed in order to clone the virus before its production, and to separate
viral sub-populations. For that, we successively inoculated diluted viral supernatant at a dilution
factor of 10 on A. castellanii. End point dilution was assessed for ﬁve days, and the lysis was controlled
by electron microscopy and ﬂow cytometry.
2.3. Cytometry Applications
After lysis detection, we managed to identify the lytic agent by applying the protocol described
by Bou Khalil et al. [12] using SYBR Green I nucleic acid gel stain (Molecular Probes, Life Technologies,
USA) to process 10 µL of the culture supernatant through ﬂow cytometry. This technique enabled us
to differentiate putative amoebal lytic agents based on previous gating of known giant viruses already
characterized by ﬂow cytometry. Once detected, and in the case of viral mixture, the end point dilution
and centrifugation methods failed to separate and purify each viral population. Therefore, we applied
a new sorting technique adapted in our lab and under review to purify Cedratvirus by ﬂow cytometry
using a BD FACS JAZZ® sorter (BD biosciences, Rungis, France) [13]. Purity control was assessed
using electron microscopy.

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Viruses 2016, 8, 300 3 of 11
With respect to the production and the puriﬁcation, brieﬂy, 15 infected ﬂasks of 150 cm2 (Corning®,
NY, USA) were pelleted using the Beckman coulter® centrifuge Avanti®J-26 XP (Beckman, France) at
14,000× g for 30 min [9]. A 25% sucrose gradient was used for the ﬁnal puriﬁcation step. After ﬁnalizing
the production, we proceeded to genome sequencing.
2.4. Electron Microscopy and Infectious Cycle Description
Negative staining was performed on the ﬁxed supernatant from co-culture. We deposited 5 µL
onto the glow-discharged grid for 20 min at room temperature. The dried grid was contrasted with a
small drop of 1% ammonium molybdate for 10 seconds and the grid was then observed on a Tecnai
G20 (FEI, Germany).
For the infectious cycle description, a pure viral suspension was used to infect four ﬂasks of
30 mL, each containing amoeba suspension of 5.105 amoebas/mL, at a multiplicity of infection (MOI)
of 10. After 30 min of post infection incubation, the amoeba monolayer was washed three times with
PAS buffer to eliminate non-internalized viruses. This time point was designated as H0. A total of
10 mL of the infected cultures were distributed into new culture ﬂasks incubated at 30 ◦C. A culture
ﬂask containing a non-infected ﬂask of amoeba was used as the negative control. At 0, 2, 4, 6, 8,
10, 12, 16, 20, 24, and 28 h post-infection (hpi), each culture ﬂask corresponding to a speciﬁc time
point was centrifuged at 720× g for 10 min, and the pellets were ﬁxed for the transmission electron
microscopy procedures. With regards to these, A. castellanii-infected cells were recovered and pelleted
for 10 min at 5000× g. The pellet was re-suspended in 1 mL of phosphate-buffered saline (PBS)
with 2% glutaraldehyde–0.1 M cacodylate and incubated for at least 1 h at 4 ◦C. Each pellet was
then washed three times with 0.1 M cacodylate–saccharose and resuspended in the same buffer.
After re-pelleting, each sample was then embedded in Epon resin by using the following standard
method: 1 h of ﬁxation in 1% osmium tetroxide, two washes in distilled water, dehydration in
increasing successive ethanol concentrations (30%, 50%, 70%, 96%, and 100% ethanol), and embedding
in Epon-812. Ultrathin (70 nm) sections were post-stained with 5% uranyl acetate and lead citrate [14].
Electron micrographs were obtained on a Tecnai G20 F20 TEM (FEI, Germany) operated at 200 keV.
ImageJ (https://imagej.nih.gov/ij/) software was used to determine particle size at the different time
points of the cycle.
2.5. Genome Sequencing
The genomic DNA of Cedratvirus was sequenced using the MiSeq Technology (Illumina Inc.,
San Diego, CA, USA) with two methods paired-end with the Nextera XT DNA sample prep kit
(Illumina), and mate pair with the Nextera Mate Pair sample prep kit (Illumina). The extracted
genomic DNA was quantiﬁed by the Qbit DNA HS Assay kit (Life technologies, Carlsbad, CA, USA)
at 513.3 ng/µL and was barcoded in order to be pooled along other projects.
To prepare the paired-end library, dilution was performed to obtain an input of 1 ng of genomic
DNA. The “tagmentation” step fragmented and tagged the DNA, and then limited-cycle polymerase
chain reaction (PCR) ampliﬁcation (12 cycles) completed the tag adapters and introduced dual-index
barcodes. The library proﬁle was validated on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.,
Santa Clara, CA, USA) with a DNA High sensitivity LabChip, and the fragment size was estimated to
be approximately 0.5 kb. After puriﬁcation on AMPure XP beads (Beckman Coulter Inc., Fullerton, CA,
USA), the libraries were normalized on speciﬁc beads according to the Nextera XT protocol (Illumina).
Normalized libraries were pooled for sequencing on the MiSeq. Automated cluster generation and
paired-end sequencing with dual index reads were performed in a 2 × 250-bp run. Total information
of 9.0 Gb was obtained from a 1019 k/mm2 cluster density, with cluster passing quality control ﬁlters
of 90.2% (17,374,744 passed ﬁltered clusters). Within this run, the index representation for Cedratvirus
was determined to be 3.85%. The 669,188 paired-end reads were trimmed and ﬁltered according to the
read qualities.

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Viruses 2016, 8, 300 4 of 11
The mate pair library was prepared with 1.5 µg of genomic DNA using the Nextera mate pair
Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate
pair junction adapter. The proﬁle of the fragmentation was validated on an Agilent 2100 Bioanalyzer
(Agilent Technologies Inc., Santa Clara, CA, USA) with a DNA 7500 LabChip. The optimal size of
obtained fragments was 6.13 kb. No size selection was performed and 631.9 ng of tagmented fragments
were circularized. The circularized DNA was mechanically sheared to generate small fragments
with an optimal size of 970 bp on the Covaris S2 device in T6 tubes (Covaris, Woburn, MA, USA).
The library proﬁle was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc.,
Santa Clara, CA, USA) and the ﬁnal concentration library was measured at 34.04 nmol/L. The library
was pooled with 11 other projects normalized at 2 nM; this pool was denatured and diluted at 15 pM
before sequencing in a 2 × 250-bp run. The total information of 7.9 Gb was obtained from a 863 K/mm2
cluster density, with cluster passing quality control ﬁlters of 94.0% (15,627,076 passed ﬁlter clusters).
2.6. Genome Assembly
Mate pair and paired-end reads were trimmed using CLC Genomics Workbench v7.5
(http://www.clcbio.com/blog/clc-genomics-workbench-7-5/). De novo assembly of 1,519,052 reads
was done using 64 word size and 50 bubble size parameters. We obtained one contig of 423,175 base
pairs (bp) and a second contig of 163,805 bp. To ﬁll gaps, speciﬁc PCR primers were designed by using
Primer-BLAST [15]. We tried the assembly with a different k-mer parameter, more adapted to the size
of our reads. Successively, we used 80 and 92 k-mer sizes with the Abyss program [16] and made a
scaffolding using SSPACE software [17] on each result. With these two assembly methods and Sanger
sequencing products we obtained a single scaffold.
2.7. Study of the Genome Organization and Genome Annotation
Repeats in the genome were investigated by complementarities from EMBOSS software
(http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome) with 200 nucleotides for maximum
length of palindromic sequences and by CRISPRFinder [18] with standard parameters. Open reading
frames were predicted by GeneMarkS [19] and the genome was deposed under BioProject
No. PREJ15450 in the EMBL-EBI database. The tRNA genes were searched for by using tRNAscan-SE
and ARAGORN softwares [20,21]. Predicted proteins of less than 50 amino acids in length were
discarded. The remaining proteins ranging from 50 to 99 amino acids in length were discarded if
they did not have a hit in the BLASTp search in the NCBI GenBank non-redundant protein sequence
database. Predicted proteins longer than 99 amino acids were kept for the analysis. A BLASTp search
against the NCBI non-redundant protein sequence database and against predicted proteins from
P. massiliensis was performed. Homology was considered signiﬁcant if the e-value was lower than
1 × 10−3. A BLASTp search was also computed with the same parameters against the clusters of
orthologous groups of proteins of the nucleocytoplasmic large DNA virus (NCVOGs) [22]. A cladistic
representation based on the presence/absence of each NCVOG was performed by MultiExperiment
Viewer (MeV) [23]. Conserved domains and putative functions of viral proteins were predicted by
comparative genomics through a BLASTp search against the NCBI non-redundant protein sequence
database and by using InterPro v58.0 (https://www.ebi.ac.uk/interpro/); results merged with speciﬁc
Delta-BLAST results [24]. We explored bona ﬁde orthologous genes by using Proteinortho v4 [25]
with 50% coverage and 30% amino acid identity and an e-value of 1 × 10−2 as signiﬁcance thresholds.
Paralogous genes were predicted by the BLASTClust program [26] with 70% coverage and 30% amino
acid identity as thresholds.
2.8. Phylogenetic Analyses
The MUSCLE program [27] was used to align amino acid sequences. The FastTree program [28]
was computed with standard parameters (using the Jones-Taylor-Thornton (JTT) model for amino

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Viruses 2016, 8, 300 5 of 11
acid substitution) by using the maximum likelihood method with 1000 bootstrap replicates. Then,
phylogenetic trees were visualized by using iTOL v3 online [29].
3. Results
3.1. Isolation of Cedratvirus
Lysis was monitored using light microscopy and cytopathic effect was observed in four wells.
The wells that presented lysis were controlled by sampling 40 µL from the supernatant on agar plates in
order to exclude bacterial contaminations. In the end, no bacterial growth was observed after ﬁve days.
Flow cytometry presumptive identiﬁcation was done on the lysis wells supernatant. We followed our
protocol based on the gating strategy [12] and we detected the presence of a mix of a mimivirus and
new unidentiﬁed particles population in one sample. The negative staining performed on this sample
culture supernatant revealed the presence of new doubled cork shaped particles corresponding to the
newly named Cedratvirus. After the failure of both centrifugation and end-point dilution methods,
and in order to obtain a pure viral population containing only Cedratvirus, we applied a new sorting
method adapted in our lab and under revision to sort the pure population of Cedratvirus. The pure
sorted fraction was re-cultured for production and sequencing.
3.2. Ultrastructure and Replicative Cycle of Cedratvirus
The Cedratvirus virions have a length ranging from 1 µm to 1.2 µm, and a maximal diameter
of 0.5 µm. The replicative cycle starts with the internalization of the viral particle by phagocytosis.
The monolayer surrounding the virion displays two variable levels of thickness at different infection
time points. In the early period of infection (Figure 1a–c), the layer measures only 40 ± 5 nm (n = 10),
then reaches 55 ± 5 nm (n = 12) (Figure 1d–i) at the stage of mature virions. This variation may
be a result of the phagosomal process of the virions in phagosomes post-internalization, and their
progressive degradation in the vacuoles after the injection of the genomic DNA into the amoebal
cytoplasm. Then, we also observed the same channel for the DNA as previously described for
P. sibericum (Figure 1a,b). Empty particles could be seen after the DNA injection and we were able to
detect particles with two-sided corks, but only one cork responsible for the DNA delivery (Figure 1c).
The virus has an infectious cycle typical for giant viruses regarding the eclipse phase, with no viral
particles detected at 4 hpi and the presence of a virus factory. Six to eight hpi, mature virions are
well-detected inside amoebas, with replication still on-going for some others (Figure 1d,e). Partial burst
cell started at 10 hpi and complete amoebal lysis was observed after 24 hpi, with a MOI of 10:1 viruses
per amoeba. Hence, the major differences with previously described pithoviruses are the presence of
two corks in virions (Figure 1h,i) and a smaller grid structure for both corks, compared with the one
observed in P. sibericum, as revealed by observation of transversal electron microscopy sections.
3.3. Genome Analysis
The Cedratvirus A11 genome is a double-stranded circular DNA molecule of 589,068 bp in length
with a GC% of 42.6%. At ﬁrst sight (Table 1), the genome appears smaller, by approximately 21 and
97 kilobase pairs (kbp) compared with P. sibericum and P. massiliensis, respectively. However, uneven
read distribution and abnormally large insert as described for P. sibericum linear genome version were
not observed for the Cedratvirus genome. In addition, no large palindromic repeats were identiﬁed
by EMBOSS Palindrome. Nevertheless, the 20 gaps of the Cedratvirus draft genome were ﬂanked by
similar short sequences. The CRISPRFinder program detected 27 areas of repeats in the genome.

7.
Viruses 2016, 8, 300 7 of 11
whereas 30.8% (177 proteins) had no signiﬁcant hit, thus being classiﬁed as encoded by ORFan genes
with an average length of 556 ± 247 nucleotides. The 397 proteins with known homologs showed
the greatest homologies with proteins from 258 viruses, 108 eukaryotes and only 31 prokaryotes.
As much as 84.1% of the best viral hits were with pithoviruses, with 107 proteins (40% of the best
viral hits) and 116 proteins (43%) having best matches with P. sibericum and P. massiliensis, respectively.
A total of 44 proteins had their best homolog in the genomes of other giant viruses, in particular,
10 with marseilleviruses. A total of 62% of the 108 best eukaryotic hits were from Micromonas pusilla
strain CCMP1545 (40 proteins, or 37%, of the eukaryotic hits), A. castellanii str. Neff (13 proteins,
or 12%) and Ectocarpus siliculosus (14 proteins, or 13%). Even if it is not rare to ﬁnd a large proportion of
protist-related genes in giant viruses, the numbers found for the two green algae were increased here by
a high number of paralogs as conﬁrmed by BLASTClust detection (Table S1, line 1), representing a large
family of genes with hypothetical functions and ankyrin repeats. In contrast, for A. castellanii, the high
number of best matches is probably linked with host speciﬁcity and probably results from horizontal
gene transfers, and these proteins could be beneﬁcial for the virus during infection. Only 31 proteins
of Cedratvirus had a prokaryotic homolog as a best hit, including seven from Bacillus spp. and two
from Paenibacillus spp. In addition, only 242 of the 574 Cedratvirus proteins had a predicted function
according to the comparative genomic analyses.
Various genes from our annotated gene set seem to be related to new, unclear metabolic processes,
for example to rhamnose synthesis. However, no complete pathway was identiﬁed. In addition,
many Cedratvirus gene functions are new among giant viruses, especially among pithoviruses.
For instance, aromatic amino acid synthesis genes were found, including an endoribonuclease
L-PSP/chorismate mutase-like encoding gene (locus tag BQ3484_354) without a homolog in other DNA
viruses, while a D-3-phosphoglycerate dehydrogenase, type 2 gene (BQ3484_10) had distant homologs
in some Chlorella viruses (a D lactate deshydrogenase). In addition, many glycosyltransferases,
acylCoA-transferases, and transferases were present in pithoviruses, but were more abundant in
Cedratvirus. Duplicate genes are present for RNA polymerase II subunit 1, as in P. sibericum
and P. massiliensis, while Cedratvirus also has two copies of genes annotated as ribonuclease III
(BQ3484_182 and BQ3484_555). Cedratvirus has one gene that has a ribonuclease H (BQ3484_454) as a
distant homolog.
3.4. Cedratvirus, Pithovirus sibericum and Pithovirus massiliensis: Three Members of a Putative
Extended Family
Analysis of the best reciprocal hits (Figure 2) for the gene repertoires of Cedratvirus and the two
previously described pithoviruses revealed that Cedratvirus shares about 21% of its gene content with
these two pithoviruses, representing 121 core genes. A total of 52.9% of this core genome consists
of ankyrin repeat-containing proteins (eight sequences) and hypothetical proteins (56 sequences).
We also noted the presence in the core genome of essential genes involved in the replication (i.e., DNA
polymerase, DNA-dependent RNA polymerase subunits, helicases) and of genes associated with DNA
reparation (i.e., DNA repair exonuclease, alkylated DNA repair protein).
Phylogenetic reconstructions based on four core genes, namely, the DNA polymerase B family,
the DNA-dependent RNA polymerase II subunits 1 and 2, and the VV-A18 helicase, revealed that
Cedratvirus is a close relative of the other pithoviruses, which raises questions about its classiﬁcation as
a new member of a putative Pithoviridae family (Figure 3 and Figures S1–S3). Congruently, the cladistic
representation based on the presence/absence of NCVOGs in the gene contents showed the relationship
between Cedratvirus and previously described pithoviruses (Figure S4).

9.
Viruses 2016, 8, 300 9 of 11
Regarding the NCVOG core genes, we found some conserved genes present also in some
other nucleocytoplasmic large DNA viruses i.e D5 helicase-primase (NCVOG0023), topoisomerase II
(NCVOG0037), mRNA capping enzyme (NCVOG1117), ribonucleotide reductase (NCVOG1353 and
NCVOG0276) and a divergent major capsid protein (NCVOG0022). However, some genes could not be
found such as the A32-like packaging ATPase (NCVOG0249), which was also absent in pithoviruses.
Finally, Cedratvirus will bring in new categories in the NCVOG gene set.
4. Discussion
Cedratvirus is a new, extraordinary giant virus. Its remarkable features include its morphological
properties, the size of its virions, the two cork virions compared to one cork in previously described
pithoviruses, and also its gene content with a high proportion of ORFans (about one third). It also has
greater coding density and GC% compared to these pithoviruses, and a large set of 449 new genes, not
detected in P. sibericum and in P. massiliensis, even when we used a low value for signiﬁcance threshold.
Moreover, 58% of the gene content of Cedratvirus consists of hypothetical proteins, representing
unknown functions. In our current understanding, Cedratvirus is the ﬁrst virus with two apertures in
its virions. We are not yet able to determine the exact capacities or beneﬁts this double-cork might
offer during replication. Regarding data from phylogenomic analyses, Cedratvirus has a genome
similar in size to the genomes of the two pithoviruses, but the gene contents are clearly different in
many instances. Moreover, the genome organization of Cedratvirus does not include the same large
palindromic repeats in the intergenic regions as observed in P. sibericum. Phylogeny reconstructions
showed that Cedratvirus is most closely related to pithoviruses, albeit distantly, but this relies on
a very limited set of highly conserved genes shared by Cedratvirus, pithoviruses and some other
giant viruses.
In a recent review [11] we discussed the characteristics of concomitant worldwide isolates of
different giant viruses in the same sample. Here, the end-point dilution and centrifugation methods
failed to isolate a pure strain of Cedratvirus. We observed again a ﬁtness propagation in favor of the
mimivirus replication. Therefore, the FACS sorter is more sensitive for detection and more efﬁcient in
the puriﬁcation and cloning process, at least in the case of a viral mixture with different ﬁtness where
it is important to sort and isolate the virus of slower replication.
Fisher recently proposed to classify giant viruses using two other genes that encode a MutS
protein and an asparagine synthetase [30], but neither Cedratvirus nor pithoviruses possess these genes.
When considering members of the four distinct lineages described in Marseilleviridae, the viral family
was found to be the closest to pithoviruses [31,32]. Some degrees of genomic conservation, and highly
similar GC%, are noted between the Marseilleviridae members. In contrast, Cedratvirus and pithoviruses
appear to be more genetically divergent. Thus, differences in their gene content is comparable to those
described between Phaeocystis globosa virus PgV-16T [33], Megavirus chiliensis, and Cafeteria roenbergensis
virus (CroV) (PgV-16T and CroV viruses are currently considered as composing an extended family
of the Mimiviridae). Indeed, about one-third of the genes of PgV-16T were not found in M. chiliensis
and CroV. Therefore, in accordance with the description for mimiCOGs by Yutin et al. [34], there is a
similar level of divergence between our results and what has been observed in the Mimiviridae family
between giant amoeba mimiviruses and distantly related viruses infecting marine eukaryotic hosts.
Furthermore, the presence/absence patterns of genes conserved among giant viruses also indicate that
Cedratvirus is most closely related to pithoviruses.
Taken together, the culture, genomic and morphological data suggest that Cedratvirus is most
closely related to pithoviruses. However, Cedratvirus is a new TRUC (Things Resisting Uncompleted
Classiﬁcation). The strength of genetic links with these viruses and a putative Pithoviridae family needs
to be clariﬁed in further studies, possibly with new additional closely-related members. Nevertheless,
the characterization of Cedratvirus broadens the knowledge of the diversity of giant amoeba viruses.
It indicates that an improved set of criteria is needed for a clear deﬁnition of genera and families
in giant viruses. The characterization of other close virus relatives of Cedratvirus, P. sibericum and